Sitosterolemia is a rare disease, only about 80 cases of which have been reported worldwide (see data from Office of Rare Diseases Research, National Institute of Health). It is an inherited lipid metabolic disorder mainly occurring in growing children by genetic modification, characterized by hyperabsorption of phytosterols such as sitosterol, campesterol and stigmasterol in the small intestine and significantly decreased biliary excretion of dietary sterols in the liver leading to accumulation of the phytosterols in the body. Thus, blood concentrations of sitosterol and stigmasterol in sitosterolemia patients are 10 to 25 times higher than in normal individuals [J. Lipid Res., 1992, 33: 945-955], leading to hypercholesterolemia or xanthoma and early atherosclerosis.
However, there is no specific treatment for sitosterolemia except taking drugs such as statin inhibiting cholesterol synthesis or ezetimibe inhibiting cholesterol absorption, and dietary treatment reducing the intake of foods rich in phytosterols is common. There is a risk that sitosterolemia may be misdiagnosed as child hypercholesterolemia or atherosclerosis with immunologic methods generally used in hospitals because structures of phytosterols are very similar with that of cholesterol. Accordingly, accurate and prompt diagnosis is of utmost importance to discriminate sitosterolemia from hypercholesterolemia or atherosclerosis. Accordingly, accurate diagnosis using precise analyzing methods such as chromatography-mass spectrometry is in demand, but there is inconvenience that a large amount of blood should be collected and the blood should be separated into serum or plasma by centrifugation when measuring the blood concentrations of phytosterols.
The dried blood spot has been mainly used for newborn screening and diagnosis of metabolic disorders [Rev. Mde. Virol., 2006, 16: 385-392; Biomed. Chromatogr. 2010, 24: 49-65]. The blood spot needs only a very small amount of blood. Accordingly, the dried blood spot has advantages of a simpler sample collection and an easier storage or transfer. However, when using the blood spot, it is difficult to determine the exact amount of the collected sample used in a test. Accordingly, because the absolute concentration of metabolites in the blood spot cannot be measured, standardized discriminating techniques, for example, evaluating metabolic ratios between compounds after measuring concentrations of all compounds exhibiting metabolic correlations, are required.
Considering the above, the present inventors tried to accomplish a quick and accurate method for discriminating sitosterolemia by metabolic ratios between compounds, which are obtained by simultaneously measuring concentrations of cholesterol metabolism-related compounds as well as phytosterols such as sitosterol, stigmasterol and campesterol, contained in the blood spot, using gas chromatography-mass spectrometry.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this disclosure and the state of the art to which this disclosure pertains.